Process for the preparation of bile acid derivatives

ABSTRACT

A process for the preparation of the compounds of general formula (I)                    
     in which 
     R 1  is H or OH; 
     R 2  is H, α-OH, or β-OH; and 
     R 3  is a straight or branched C 1 -C 4  alkyl group or a benzyl group, 
     comprising the reduction of compounds of formula (III)                    
     wherein R 1 , R 2  and R 3  have the same meanings as in formula I, in the presence of sodium borohydride.

The present invention relates to a novel process for the preparation ofbile acid derivatives in which an amino group is present at the 3βposition.

The most important bile acids are represented in the following scheme 1:

More particularly, the invention relates to a process for thepreparation of compounds of formula (I) from compounds of formula (II):

in which

R₁ is H or OH;

R₂ is H, α-OH, or β-OH; and

R₃ is a straight or branched C₁-C₄ alkyl group, or a benzyl group.

The compounds of general formula (I) are important intermediates for thepreparation of compounds useful for a number of pharmaceuticalapplications. They are, for example, used for the preparation ofinhibitors of bile acids intestinal absorption (see EP-A-0489423) or ascarriers for active compounds in the enter-hepatic circulation (seeEP-A-0417725).

Last, but not least, is the use thereof for the preparation of contrastagents for medical diagnosis using Magnetic Resonance, such as thosedescribed in WO-A-95/32741, resulting from the conjugation of a bileacid with a chelating agent, which are capable of chelating the ions ofparamagnetic bi- and trivalent metals, in particular the gadolinium ion,or in the publication: Anelli P. L. et al., Acta Radiologica, 38, 125,1997.

EP-A-614,908 discloses the preparation of the derivatives of generalformula (I), comprising the following steps:

a) formation of phthalimido derivatives of general formula (III) byreacting compounds of general formula (II) with phthalimide;

b) treatment of compounds of general formula (III) with hydrazinehydrate or phenylhydrazine;

c) subsequent treatment of the reaction products from step b) withmineral acids to form the addition salts, and

d) liberation from the salt to give the compounds of general formula(I).

As already widely discussed in EP-A-614,908, the formation of thephthalimido derivative is carried out by means of the well knownMitsunobu reaction (see Synthesis, 1, 1981; Org. React. Vol. 42, 335(1992)), namely in the presence of a suitable phosphine and DEAD(diethylazodicarboxylate) or DIAD (diisopropylazodicarboxylate), in anorganic solvent such as dioxane or tetrahydrofuran, at a temperaturewhich ranges from 20 to 50° C. The Mitsunobu reaction yields the finalproducts with inversion of configuration.

The process illustrated makes use of hydrazine hydrate orphenylhydrazine, particularly dangerous products due to theirascertained cancerogenicity.

It has now surprisingly been found that the reduction of the phthalimidogroup in the compounds of formula (III) can be advantageously carriedout with sodium borohydride.

It is therefore the object of the present invention a process for thepreparation of the compounds of general formula (I) comprising thereduction reaction of the compounds of general formula (III) in thepresence of sodium borohydride, according to the following Scheme 2:

in which

R₁ is H or OH;

R₂ is H, α-OH, or β-OH; and

R₃ is a straight or branched C₁-C₄ alkyl group, or a benzyl group.

A further object of the present invention is the process for thepreparation of the compounds of general formula (I) comprising thereduction of the compounds of general formula (III) in the presence ofsodium borohydride, through formation of the novel compounds of generalformula (IV), and subsequent deprotection by treating said compoundswith acids, according to the following Scheme 3:

in which R₁, R₂ and R₃ have the meanings defined above.

Compounds of general formula (IV) are novel, useful intermediates in theprocess according to Scheme 3 and their recovery will be described inthe Experimental Section.

Particularly preferred is the process for the preparation of compoundsof formula (Ia), according to Scheme 3, starting from compounds offormula (IIIa), which are derivatives of cholic or deoxycholic acid,

in which

R₃ has the meanings defined above and

R₄ can be a hydrogen atom or a hydroxy group.

A further object of the present invention is the process for thepreparation of compounds of formula (Ib), according to Scheme 4,starting from compounds of formula (IIIb), which are deoxycholic acidderivatives,

in which R₃ has the meanings defined above.

Particularly preferred is the process for the preparation of compound(Ic), according to Scheme 5, starting from compound (IIIc), adeoxycholic acid derivative,

The compounds of formula (IIIc) and (IVc), (3β, 5β,12α)-3-[1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)]-12-hydroxycholan-24-oicacid methyl ester and (3β, 5β,12α)-12-hydroxy-3-[[(2-hydroxymethyl)benzoyl]amino]cholan-24-oic acidmethyl ester, respectively, are novel and they are useful intermediatesfor the preparation of compound of formula (Ic), a deoxycholic acidderivative.

The compounds of formula (III) are synthesized according to the generalprocedure already described above and exemplified in EP-A-614,908.

Preferred reaction conditions comprise:

1) selection of a reaction temperature from 15 to 25° C., thusdecreasing the amount of diisopropylazodicarboxylate to a small excessto the stoichiometric;

2) crystallization of phthalimido derivatives (III) from MeOH instead of2-PrOH, thereby reducing the volume of the crystallization solvent by atleast four times.

The reduction of phthalimido derivatives with NaBH₄ has never beforebeen described in the field of the compounds of the present invention,but references exist in the literature, (T. W. Greene; P. G. M. Wuts“Protective groups in Organic Synthesis”; 3^(rd) Ed John Wiley and sons,New York, 1999), reporting the use of a large excess of this reducingagent (5-10 mol) to deprotect a phthalimido group.

The teaching contained in the most important paper [Osby, TetrahedronLetters, Vol. 25, 2093 (1984)] i.e. the use of 2-PrOH/H₂O=6/1 as areduction solvent proved to be definetly unsuitable for the process ofthe present invention. In fact, due to the poor solubility of compoundsof formula (III), it was necessary to operate at temperatures around 40°C. and with a 2.5% maximum concentration which is of course industriallyunacceptable.

On the other hand, Osby already evidenced that the proposed conditionswere not particularly suitable to deprotect phthalimido groups fromderivatives containing an ester function. Osby himself in fact observedthat the deprotection of this group in said derivatives was accompaniedby reduction and hydrolysis of the ester group with consequent decreasein reaction yields.

The Applicant found that when reducing the compounds of formula (III)under the Osby conditions, compounds of formula (I) were recovered inaround 65% yields, but contaminated by remarkable amounts ofby-products, mainly the product from the reduction of the ester group at24- to alcohol. This phenomenon is still significant even when markedlyreducing the NaBH₄ excess. In order to better understand the genesis ofthe by-products, the reaction carried out under the Osby conditions wasworked up at the end of the reduction with NaBH₄ and before the acidhydrolysis and the side-products recovered by silica chromatography hadthe formulae (V)-(VIII). These by-products are of course the precursorsof the impurities evidenced in compounds of formula (I) and account forthe low reaction yields.

In the Experimental section the recovery and the characterization of thevarious intermediates, in particular in the case of deoxycholic acid,will be reported.

Compounds of formula (IV) are substantially inseparable from compounds(V) by crystallization from different solvents. Not even the conversionof compounds (IV) into the corresponding acids of formula (VI) allowsthe elimination of the by-products of formula (V).

It has surprisingly been found that using dipolar aprotic solvents suchas DMA (dimethylacetamide), DMF (dimethylformamide), DMSO(dimethylsulfoxide), NMP (N-methyl-pyrrolidone), in place of analcoholic solvent, such as isopropanol described by Osby, these problemscould be overcome. Particularly preferred are dimethylacetamide andN-methylpyrrolidone.

Using said solvents the concentration of compounds (III) could beincreased up to more than 10%, without increasing the amount ofside-products.

Moreover, under the conditions of the present invention, the NaBH₄excess could be decreased, compared with what reported above.

In particular, the molar equivalents of reducing agent can be loweredfrom a strong excess (about 5) to substantially stoichiometric values(1.2-0.85) to the substrate, without adversely affecting the reactionyields.

A further aspect of the present invention is the use, simultaneouslywith the dipolar aprotic solvents, of a buffer solution at pH 7.5-9which allows to control pH during the reaction. This allows to inhibitthe hydrolysis of sensitive functions minimizing the formation ofby-products (VI)-(VIII).

The compounds of formula (IV) are then transformed into those of formula(I) by treatment in dipolar aprotic or alcoholic solvent, optionally inmixture with water, and in the presence of a mineral or organic acid(such as acetic acid).

Particularly preferred is the use of HCl in methanol solution. Thesubsequent liberation of the free base from the salt is carried out bytreatment of the solution in one of the above cited solvents withaqueous bases (such as NaOH, Na₂CO₃. . . ).

As already cited above, compounds of formula (I) are useful for thepreparation of medicaments for decreasing cholesterol plasmatic levels,(EP-A-417,725 or EP-A-489,423) or of contrast agents for the nuclearmagnetic resonance diagnosis, as described in WO-A-95/32741.

For the latter, the known synthesis comprised the transformation ofcompounds (II) into the corresponding compounds (I) by intermediateformation of the azide at the 3β position, according to Mitsunobureaction, as represented in the following Scheme 6:

This Synthetic scheme is unsuitable for scaling-up, being azidespotentially dangerous and the key reagent (diphenylphosphoryl azide)extremely expensive.

The present invention relates to a process for the preparation ofchelating agents of general formula (IX), capable of chelatingparamagnetic bi-trivalent metal ions, selected the group consisting ofFe⁽²⁺⁾, Fe⁽³⁺⁾, Cu⁽²⁺⁾, Cr⁽³⁺⁾, Gd⁽³⁺⁾, Eu⁽³⁺⁾, Dy⁽³⁺⁾, La⁽³⁺⁾, Yb⁽³⁺⁾or Mn⁽²⁺⁾,

in which

R₁, and R₂ have the meanings defined above;

R₅ is a hydrogen atom or a (C₁-C₅) alkyl group unsubstituted orsubstituted with a carboxylic group;

X is the residue of a polyaminocarboxylic ligand and of derivativesthereof, selected from the group consisting of:ethylenediaminotetraacetic acid (EDTA), diethylenetriaminopentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (HPDO3A),4-carboxy-5,8,11-tris(carboxy-methyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oicacid (BOPTA);

L is a residue of formula

m is an integer from 1 to 10, wherein for values above 1, the values ofp and q can differ in the monomeric units;

q is 0 or 1;

p can range from 0 to 10, p and q not being at the same time zero,

said process comprising the following steps:

a) formation of compounds (III) starting from compounds (II) by reactionwith phthalimide according to Mitsunobu's procedure, at temperaturesranging from 15 to 25° C., in the presence of an azodicarboxylateselected from DEAD (diethylazodicarboxylate) or DIAD(diisopropylazodicarboxylate) in amounts ranging from 1.1 to 1.3 molarequivalents, in a solvent selected from the group consisting of THF,dioxane, toluene and DMF;

b) reduction of compounds (III) with NaBH₄ to give compounds (IV);

c) acidic hydrolysis of compounds (IV) followed by neutralization togive compounds (I);

d) condensation of compounds (I) with the reactive residues of thepolyaminocarboxylic ligands defined above.

Particularly preferred is the process for the preparation of compounds(IXa), which are cholic or deoxycholic acid derivatives, according tothe following Scheme 7:

in which R₃, R₄ and R₅ have the meanings defined above.

Furthermore, particularly preferred is the process for the preparationof compounds (X), in which in formula (IXa) the residue X is DTPAsubstituted on the chain at the central position, R₆ can be a hydrogenatom or a carboxylic group and the L chain, R₄ and R₅ have the meaningsdefined above.

Particularly preferred is the process for the preparation of compounds(Xa)

in which the L chain, R₄ and R₅ have the meanings defined above.

Furthermore, particularly preferred is the process for the preparationof the following novel compound, of general formula (Xa):

[3β(S), 5β,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid;

Other compounds belonging to this class, whose preparation has alreadybeen described in WO-A-95/32741, are the following:

[3β(S),5β, 7α,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;

[3β(S),5β, 7α,12α]-3-[[4-[[5-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]amino]-7,12-dihydroxycholan-24-oicacid.

It is moreover preferred the process for the preparation of thefollowing compounds of formula (Xb), in which in formula (X) R₆ is ahydrogen atom, and R₄, R₅ and L have the meanings defined above.

Also preferred is the process for the preparation of the following novelcompound, belonging to the class of general formula (Xb):

(3β, 5β, 7α,12α)-3-[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

Other compounds belonging to this class, already described inWO-A-95/32741, are the following:

(3β, 5β, 7α,12α)-3-[[[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;

(3β, 5β, 7α,12α)-3-[[6-[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-1-oxohexyl]amino]-7,12-dihydroxycholan-24-oicacid.

Particularly preferred is also the process for the preparation ofcompounds of formula (XI), in which in formula (IXa) the residue Xderives from DTPA, and R₄, R₅ and L have the meanings defined above.

Other compounds belonging to this class, whose preparation was alreadydescribed in WO-A-95/32741, are the following:

(3β, 5β, 7α, 12α)-3-[[N-[N-[2-[[2-[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]ethyl]-N-(carboxymethyl)glycyl]glycyl]amino]-7,12-dihydroxycholan-24-oicacid;

18-[[(3β, 5β, 7α,12α)-23-carboxy-7,12-dihydroxy-24-norcholan-3-yl]amino]-3,6,9-tris(carboxymethyl)-11,18-dioxo-3,6,9,12-tetraazaoctadecanoicacid.

Particularly preferred is also the process for the preparation of thefollowing compounds of formula (XII), in which in formula (IXa) theresidue X is DOTA, and R₄, R₅ and L have the meanings defined above.

Furthermore, preferred is the process for the preparation of compoundsof formula (XIII), in which in formula (IXa) the residue X is EDTA, andR₄, R₅ and L have the meanings defined above.

Particularly preferred is the process for the preparation of thefollowing compounds of formula (XIII):

[3β(S), 5β, 7α,12α]-3-[[4-[[5-[[2-[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]amino]-7,12-dihydoxycholan-24-oicacid

[3β(S) , 5β,12α]-3-[[4-[[2-[[bis(carboxymethyl)amino]ethyl](carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

[3β(S),5β]-3-[[4-[[2-[[bis(carboxymethyl)amino]ethyl](carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-oxocholan-24-oicacid

The experimental conditions used will be illustrated in detail in theExperimental Section.

Experimental Section Example 1

(3β, 5β,12α)-3-[1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)]-12-hydroxycholan-24-oicacid methyl ester

Phthalimide (202.8 g; 1.378 mol) is suspended in a solution of (3β, 5β,12α)-3,12-dihydroxycolan-24-oic acid methyl ester (prepared as describedin Steroids, vol. 37, 239) (511.9 g; 1.259 mol), and triphenylphosphine(372.7 g; 1.421 mol) in THF (1.5 L), then a solution ofdiisopropylazodicarboxylate (284.7 g; 1.408 mol) in THF (0.49 L) isdropped therein in 1.5 h, keeping the temperature at 15° C. Theresulting solution is left at room temperature for 18 h. The solvent isdistilled off and the oily residue is added with MeOH (3.2 L) andstirred for 20 h. The resulting crystalline product is filtered, washedwith MeOH (1.4 L) and dried to give the desired product (481.3 g; 0.898mol). Mother liquors and washings are concentrated to give a second cropof product (61.6 g; 0.114 mol)

Yield: 80%

m.p. : 160-162° C.

HPLC assay: 98.7%

Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.017MH₃PO₄ in water B = CH₃CN Gradient: min % A % B  0 82 18 30 15 85 45 1585 Flow: 1 mL min⁻¹ Detection (UV): 210 nm; Elemental analysis C H N %calc.: 73.99 8.47 2.61 % found: 73.96 8.51 2.62 TLC: silica gel plate60F 254 Merck Eluent: AcOEt/n-hexane = 4:6 Rf: 0.35 Detection: 2%Ce(SO₄).4 H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂ SO_(4 in water)

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Example 2

Reduction of compounds of formula (III) according to a procedure similarto that described in Osby, Tetrahedron Letters, Vol. 25, 2093 (1984).

(3β, 5β, 12α,)-3-Amino-12-hydroxycholan-24-oic acid methyl ester

The compound prepared in Example 1 (75 g; 0.14 mol) is dissolved in2-PrOH (2.5 L) at 75° C. The solution is vigorously stirred and cooledto 50° C., then added with H₂O (430 mL) to obtain a suspension which isadded with solid NaBH₄ (26.5 g; 0.70 mol) in portions during 30 min. Thereaction mixture is stirred at 40° C. for 2.5 h. CH₃COOH is carefullyadded to pH 4.5-5 and the mixture is heated at 80° C. for 24 h, keepingpH 4.5-5 by repeated additions of CH₃COOH. After cooling to roomtemperature and evaporation of the solvent, a residue is obtained whichis dissolved in water (1 L). The solution is alkalinized to pH 10 with2M NaOH to obtain a precipitate which is stirred for 1 h, then filtered,washed with water and dried.

Crystallization from acetonitrile (1.2 L) yields the desired product(36.9 g; 0.091 mol)

Yield: 65%

HPLC assay: 87%

Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.01Msodium pentanesulfonate in water buffered to pH 2.5 with H₂SO₄ B = CH₃CNGradient: min % A % B  0 82 18 30 15 85 45 15 85 Flow: 1 mL min⁻¹;Detection (UV): 210 nm;

Silica gel chromatography of the crude compound gave the main by-product(2.5 g) which was:

m.p.: 174-175° C. Elemental analysis C H N % calc.: 76.34 11.48 3.71 %found: 76.18 11.65 3.52

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

In another reaction carried out under the above described conditions, atthe end of the reduction the NaBH₄ excess was destroyed with acetic acidand the reaction mixture (about neutral pH) was evaporated and taken upwith water to obtain a crude which was purified by silica gelchromatography. Together with the expected compound (IVb) (see EXAMPLE3) remarkable amounts of the by-products, reported hereinbelow togetherwith their analytical characterization, were recovered. This widelyaccounts for the low recovery yield of compound (Ib) after hydrolysiswith acetic acid at 80° C.

N-[(3β, 5β, 12α)-12,24-Dihydroxycholan-3-yl]-2-(hydroxymethyl)benzamide

m.p.: 200-203° C. Elemental analysis C H N % calc.: 75.11 9.65 2.74 %found: 74.66 9.61 2.71

(3β, 5β,12α)-12-Hydroxy-3-[[2-(hydroxymethyl)benzoyl]amino]cholan-24-oic acid

m.p.: >260° C. Elemental analysis C H N % calc.: 73.11 9.01 2.66 %found: 73.32 9.15 2.76

N- [(3β, 5β,12α)-12-Hydroxy-24-methoxy-24-oxocholan-3-yl]-2-(hydroxymethyl)benzamide

The product was characterized as ammonium salt m.p.: 156-162° C.Elemental analysis C H N % calc.: 69.44 8.83 4.91 % found: 69.27 8.894.82

(3β, 5β, 12α)-12-Hydroxy-3-[[(2-carboxybenzoyl]amino]cholan-24-oic acid

m.p. 193-196° C. Elemental analysis C H N % calc.: 71.48 8.06 2.60 %found: 71.18 8.25 2.55

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Example 3

(3β, 5β,12α)-12-Hydroxy-3-[[(2-hydroxymethyl)benzoyl]amino]cholan-24-oic acidmethyl ester

A solution of the compound prepared in Example 1 (225 g; 0.42 mol) inDMA (1.75 L), vigorously stirred at 34° C., is diluted with 2M bufferphosphate pH 8 (500 mL) obtaining a mixture at 43° C. Solid NaBH₄ (15.45g; 0.408 mol) is added in 5 min and the suspension is stirred at 47-48°C. After 55 min an almost clear solution is obtained which is added withCH₃COOH to neutralize the hydride excess and adjust pH from 12.8 toabout 8.

The mixture is poured into H₂O and stirred for 3 h, the precipitate isfiltered and suspended in H₂O then stirred for 30 min. Finally, theprecipitate is filtered, washed with H₂O and dried to give the desiredproduct (224.6 g; 0.416 mol)

Yield: 99%

m.p. : 190.2-192.7° C.

HPLC assay: 93%

Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.017MH₃PO₄ in water B = CH₃CN Gradient: min % A % B  0 82 18 30 15 85 45 1585 Flow: 1 mL min⁻¹; Detection (UV) : 210 nm; Elemental analysis C H N %calc.: 73.43 9.15 2.60 % found: 73.43 9.19 2.56 TLC: silica gel plate60F 254 Merck Eluent: AcOEt/n-hexane/MeOH = 5:5:1 Rf: 0.79 Detection: 2%Ce(SO₄).4 H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Example 4

(3β, 5β, 12α)-3-Amino-12-hydroxycholan-24-oic acid methyl esterhydrochloride

A suspension of the compound prepared in Example 3 (223.5 g; 0.414 mol)in MeOH (1.5 L) and 37% HCl (52 mL;

0.63 mol) is heated at 50° C. under stirring. After about 1.5 h asolution is obtained and after a further 2 h, the reaction is completed.The solvent is evaporated off and the residue is stirred at 60° C. for 2h with CH₃CN. After 15 h at room temperature the precipitate isfiltered, washed with CH₃CN and dried to give the desired product (183.3g; 0.414 mol).

Yield: 100%.

m.p.: >250° C.

HPLC assay: 95%

Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.01Msodium pentanesulfonate in water buffered to pH 2.5 with H₂SO₄ B = CH₃CNGradient: min % A % B  0 82 18 30 15 85 45 15 85 Flow: 1 mL min⁻¹;Detection (UV) : 210 nm;

Example 5

(3β, 5β,12α,)-3-Amino-12-hydroxycholan-24-oic acid methyl ester

The compound prepared in Example 4 (89.5 g; 0.202 mol) is dissolved inMeOH (460 mL) at 55° C., then the solution is cooled to about 40° C. andpoured, under strong stirring, into H₂O while adding solid Na₂CO₃ tokeep pH 9.8. After stirring for 2 h, the suspension is filtered, thesolid is suspended in H₂O and stirred for 2 h, filtered and washed withH₂O. After drying, the desired product is obtained (74.9 g; 0.185 mol).

Yield: 91%

m.p. : 155-155.5° C.

HPLC assay: 96.5%

Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.01Msodium pentanesulfonate in water buffered to pH 2.5 with H₂SO₄ B = CH₃CNGradient: min % A % B  0 82 18 30 15 85 45 15 85 Flow: 1 mL min⁻¹Detection (UV): 210 nm; GC assay: 96.3% Stationary phase: PE 1 (OV 1)Film thickness: 0.25 μm Column (WCOT): 30 m × 0.32 mm Startingtemperature in the oven: 255 ° C., after 5 min the temperature isincreased by 0.6° C./min to reach 290° C. Injector temperature: 290° C.Temperature FID: 290° C. Elemental analysis C H N % calc.: 74.03 10.693.45 % found: 73.91 10.78 3.44 TLC: silica gel plate 60F 254 MerckEluent: CHCl₃/MeOH/25% NH₄OH = 6:2:0.5 Rf: 0.69 Detection: 2% Ce(SO₄).4H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Compounds of formula (I), whose formulae and analytical data arereported hereinbelow, were prepared analogously:

m.p. (° C.) Rf^(a)

164- 166 0.48

121- 123 0.67

127- 128 0.80 ^(a)TLC: silica gel plate 60F 254 Merck Eluent:CHCl₃/MeOH/25% NH₄OH = 6:2:0.5 Detection: 2% Ce(SO₄).4H₂O, 4.2%(NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

The ¹H-NMR, ¹³C-NMR, IR and MS spectra as well as the elementaryanalysis are consistent with the indicated structure.

Example 6

[3β(S), 5β,12α]-3-[[4-[Bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

A) [3β(S), 5β,12α]-3-[[4-[Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]-5-(1,1-dimethylethoxy)-1,5-dioxopentyl]amino]-12-hydroxycholan-24-oicacid methyl ester

Triethylamine (2.23 g; 22 mmol) is added to a solution of 8.93 g of [3β,5β, 12α]-3-amino-12-hydroxycholan-24-oic acid methyl ester (prepared inExample 5) (22 mmol), 16.41 g ofN,N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid (1,1-dimethylethyl) ester (prepared as described in WO-A-95/32741:example 15) (22 mmol) and 3.91 g of diethyl cyanophosphate (24 mmol) in300 mL of DMF at 0° C. After 1.5 h at 0° C. and 18 h at roomtemperature, the reaction mixture is evaporated and the residue isdissolved in AcOEt. The solution is washed with a NaHCO₃ saturatedsolution and H₂O, dried (Na₂SO₄) and evaporated. The crude is purifiedby flash chromatography to give the desired product (20.67 g; 18.2mmol).

Yield: 83% [α]_(D) ²⁰ = −6.75 (c 2.0, CHCl₃) Elemental analysis C H N %calc.: 65.69 9.60 4.94 % found: 66.54 9.95 4.99 TLC: Carrier: silica gelplate 60F 254 Merk Eluent: n-hexane/EtOAc = 1:1 R_(f) = 0.09 Detection:Ce(SO₄ ₂.4 H₂O (0.18%) and (NH₄)₆Mo₇O₂₄.4 H₂O (3.83%) in 10% H₂SO₄

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

B) [3β(S), 5β,12α]-3-[[4-[Bis[2-[bis(carboxymethyl)amino]ethyl]amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

The compound prepared at step A) (19.72 g; 17.4 mmol) is dissolved in105 mL of CF₃CO₂H at room temperature. After 26 h the solution isevaporated and the residue is treated with H₂O; the solid is filtered,washed with H₂O and partially dried under vacuum. The resultingintermediate is dissolved in H₂O at pH 13 with 1M NaOH.

After 5 h at room temperature the solution is dropwise added with 0.5MHCl at pH 1.4. After 15 h at room temperature the precipitate isfiltered, washed with H₂O and dried under vacuum to give a crude whichis purified by chromatography on Amberlite^((R)) XAD 1600 resin toobtain the desired product (9.92 g; 11.8 mmol).

Yield: 68% m.p.: 184° C. (dec.) Compleximetric titer (0.1M GdCl₃): 99.3%Acidic titer (0.1M NaOH): 99.8% [α]_(D) ²⁰ (c 2.0; 1M NaOH) λ (nm) 589578 546 436 405 365 [α] +23.61 +24.59 +27.90 +46.67 +55.61 +71.40Elemental analysis C H N % calc.: 58.70 7.93 6.68 % found: 58.22 8.166.59 H₂O 0.70% TLC: silica gel plate 60F 254 Merck Eluent:CHCl₃/MeOH/NH₄OH = 5:4:2 R_(f) = 0.13 Detection: Ce(SO₄)₂.4 H₂O (0.18%)and (NH₄)₆Mo₇O₂₄.4 H₂O (3.83%) in 10% H₂SO₄

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Example 7

(3β, 5β, 7α,12α)-3-[[[Bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

A)N-[[Bis[2-[bis[2-(1,1-dimethylethoxy-2-oxoethyl]amino]ethyl]amino]-acetyl]glycine

6.5 g of glycylglycine (49.3 mmol) are suspended in 100 mL of a1:1=H₂O:EtOH mixture and dissolved at pH 10 with 10M NaOH (4.8 mL).N-(2-bromoethyl)-N[2-(1,1-dimethylethoxy)-2-oxoethyl]glycine-1,1-dimethylethylester (42 g; 110.9 mmol) in 40 mL of EtOH is dropped in 2 h keeping pHat 10.5 with 10M NaOH (5.8 mL). The solution quickly turns to anemulsion, which is dissolved after 2.5 h by addition of 10M NaOH. After22 h the solvent is evaporated off, the residue is diluted with waterand extracted with CH₂Cl₂. The organic phase is washed with H₂O, driedand evaporated, to give a residue which is purified by flashchromatography. The residue is dissolved in water, pH is adjusted to 4.5by addition of 1M HCl and the solution is extracted with chloroform. Theorganic phase is washed with H₂O, dried and evaporated, to give 13 g ofthe desired compound (19.3 mmol).

Yield: 39% Elemental analysis C H N % calc.: 56.95 8.66 8.30 % found:56.67 8.68 8.30 TLC: silica gel plate 60F 254 Merck Eluent:CHCl₃/MeOH/NH₄OH 25% = 6:3:1 R_(f) = 0.65 Detection: KMnO₄ in alkalinesolution

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

B) (3β, 5β, 7α,12α)-3-[[[[[Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid methyl ester

2.8 mL of TEA (20.2 mmol) are dropwise added in 5 min to a solution of13.6 g of the compound prepared at step A) (20.2 mmol), 8.52 g of (3β,5β, 7α, 12α)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester(prepared in Example 5) (20.2 mmol) and DEPC (3.4 mL; 22.2 mmol) in DMF(290 ml) stirring at 0° C. After 1 h the reaction is warmed to roomtemperature and the solution is stirred for 6.5 h. 0.3 mL of DEPC (2mmol) are added and the solution is stirred for a further 15.5 h. DMF isevaporated off, the residue is dissolved in EtOAc, washed with aq.NaHCO₃ and then water and finally dried. Purification by flashchromatography yields 13.7 g of the desired product (12.7 mmol).

Yield: 63% [α]_(D) ²⁰ = +5.26 (c 1.5; CHCl₃) Elemental analysis C H N %calc.: 63.48 9.25 6.49 % found: 63.22 9.40 6.40

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

C) (3β, 5β, 7α,12α)-3-[[[[[Bis[2-[bis(carboxymethyl)amino]ethyl]-amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

12.85 g of the compound prepared at step B) (12 mmol) are dissolved inTFA (210 mL) stirring at 0÷5° C. After 16 h TFA is evaporated off togive a residue which is dissolved in 90 mL of 0.8M NaOH at pH 13 andstirred at room temperature for 15 h. The solution is concentrated to 50mL, dropped in 105 mL of 0.6M HCl and stirred for 2 h. The solid isfiltered, washed with 0.1M HCl and dried to obtain a crude which ispurified by chromatography. The fractions containing the desiredcompound in salified form are evaporated to give a residue which isdissolved in water and dropped into 0.1M HCl at pH 1.45. The precipitateis filtered, washed with 0.1M HCl and dried to give 2.6 g of the desiredproduct (3.1 mmol).

Yield: 26% m.p.: 120-125° C.

HPLC assay: 98% (area %)

The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with the indicatedstructure.

Example 8

Preparation of cholic acid derivative chelating agents.

Using (3β, 5β, 7α, 12α)-3-amino-7,12-dihydroxycholan-24-oic acid methylester prepared as described in example 5, and following the proceduresdescribed in WO-A-95/32741, the following chelating agents wereprepared:

[3β(S), 5β, 7α,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;

[3β(S), 5β, 7α,12α]-3-[[4-[[5-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;

(3β, 5β, 7α,12α)-3-[[[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;

(3β, 5β, 7α,12α)-3-[[6-[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-1-oxohexyl]amino]-7,12-dihydroxycholan-24-oicacid;

(3β, 5β, 7α, 12α)-3-[[N-[N-[2-[2-[2-[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]ethyl]-N-(carboxymethyl)glycyl]glycyl]amino]-7,12-dihydroxycholan-24-oicacid;

18-[[(3β, 5β, 7α,12α)-23-carboxy-7,12-hydroxy-24-norcholan-3-yl]amino]-3,6,9-tris(carboxymethyl)-11,18-dioxo-3,6,9,12-tetraazaoctadecanoicacid.

What is claimed is:
 1. A process for the preparation of compounds ofgeneral formula (I)

in which R₁ is H or OH; R₂ is H, α-OH, or β-OH; and R₃ is a straight orbranched C₁-C₄ alkyl group, or a benzyl group; comprising the reductionof compounds of general formula (III)

wherein R, R₂ and R₃ have the same meanings as in formula (I), in thepresence of sodium borohydride.
 2. A process as claimed in claim 1,which comprises the preparation of compounds of general formula (IV),

in which R₁, R₂ and R₃ have the same meanings as in claim 1, and thesubsequent deprotection by acid treatment.
 3. A process as claimed inclaim 2, for the preparation of compounds of general formula (Ia),

in which R₃ has the same meanings as in claim 1 and R₄ can be a hydrogenatom or a hydroxyl group.
 4. A process as claimed in claim 3, for thepreparation of compounds of general formula (Ib),

in which R₃ has the same meanings as in claim
 1. 5. A process as claimedin claim 4, for the preparation of the compound of formula (Ic)


6. A process according to claims 1, 2, 3, 4 or 5 in which the reductionwith sodium borohydride is carried out in a dipolar aprotic solventselected from the group consisting of DMA (dimethylacetamide), DMF(dimethylformamide), DMSO (dimethylsulfoxide), NMP(N-methylpyrrolidone).
 7. A process as claimed in claim 6, in which thereduction with sodium borohydride is carried out in a dipolar aproticsolvent selected from DMA and NMP.
 8. A process according to claims 1,2, 3, 4 or 5 in which the amount of NaBH₄ is from 1.2 to 0.85 mols.
 9. Aprocess according to claims 1, 2, 3, 4 or 5 in which the reduction withsodium borohydride is carried out in a dipolar aprotic solvent and inthe presence of a buffer solution at pH 7.5-9.
 10. A process accordingto claims 1, 2, 3, 4, or 5 in which compounds of formula (IV) aretransformed into those of formula (I) by treatment in a dipolar aproticsolvent, optionally in mixture with water, and in the presence of amineral or organic acid.
 11. A process as claimed in claim 10, in whichthe mineral acid is HCl in methanol solution.